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Free keywords:
Animals
Blotting, Western
Cell Nucleus/*metabolism
Cells, Cultured
Down-Regulation
Electrophoresis, Gel, Two-Dimensional
Fluorescein-5-isothiocyanate
Fluorescent Antibody Technique, Direct
Fluorescent Dyes
Gene Expression Regulation, Neoplastic
Isoelectric Focusing
Isoenzymes/genetics/*metabolism
Leukemia, Erythroblastic, Acute/*metabolism/pathology
Mice
Microscopy, Fluorescence
Peptide Mapping
Phospholipase C beta
Precipitin Tests
Proteomics/*methods
RNA-Binding Proteins/*metabolism/physiology
*Signal Transduction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Subcellular Fractions/metabolism
Type C Phospholipases/genetics/*metabolism
Abstract:
An extensive body of evidence links inositide-specific phospholipase C (PLC) to the nucleus and the main isoform located in the nucleus is PLCbeta(1). Constitutive overexpression of nuclear PLCbeta(1) has been previously shown to inhibit Friend erythroleukemia cells differentiation and to induce cell cycle progression targeting cyclin D3. The aim of this study was to identify new proteins regulated by PLCbeta(1) overexpression, given the role exerted by its signaling in the nucleus during cell growth and differentiation. To identify novel downstream effectors of nuclear PLCbeta(1)-dependent signaling in Friend erythroleukemia cells, we performed the high-resolution 2-DE-based proteomic analysis. Using a proteomic approach we found that SRp20, a member of the highly conserved SR family of splicing regulators, was down-regulated in cells overexpressing nuclear PLCbeta(1) as compared with wild-type cells. Reduction in SRp20 was confirmed by 2-D Western blotting. Moreover, we have shown that nuclear PLCbeta(1) is bound to the SRp20 splicing factor. Indeed, by immunoprecipitation and subcellular fractioning, we have demonstrated that endogenous PLCbeta(1) and SRp20 physically interact in the nucleus. Here we show the existence of a PLCbeta(1)-specific target, the splicing factor SRp20, whose expression is specifically down-regulated by the nuclear signaling evoked by PLCbeta(1).
